SUMMARY/ABSTRACT Natural products (NPs) isolated from diverse sources stock the majority of our nation's biomedical arsenal. Due to emerging antibiotic resistance and limited cancer treatments, additional NP discoveries are of fundamental importance. Microbial genomics has unveiled `gifted' microorganisms?those capable of synthesizing many structurally diverse NPs. However, most gifted microorganisms do not synthesize NPs under laboratory conditions. Understanding elicitors that activate NP synthesis would enable the discovery of new and potentially therapeutic NPs. In select gifted microorganisms (e.g. actinobacteria), chemical and biological elicitors modulate NP production; in others (e.g. myxobacteria), the effects of chemical and biological stimuli are unknown. Three lines of evidence support a role for chemical and biological stimuli in myxobacterial NP regulation. First, myxobacteria rely on NPs for predation. Second, myxobacterial genomes are enriched for regulatory networks, such as serine-threonine protein kinases, which detect and respond to external stimuli. Third, preliminary data described herein is consistent with our hypothesis. Chemical (the sub-lethal antibiotic norfloxacin) and biological (the prey E. coli and the predator Dictyostelium descoideum AX-2) stimuli modulate known and putative novel natural products in Myxococcus xanthus DK1622. For these reasons, we hypothesize that biological and chemical stimuli modulate myxobacterial NP synthesis. The proposed project assesses the consequence of competitive stimuli on myxobacterial secondary metabolism, evaluates their bioactivity against a cancer line, and elucidates the structure of myxobacterial NPs. In Aim 1, the consequences of chemical and biological stimuli on myxobacterial secondary metabolism will be further investigated. Ecological stimuli (bi-partite competitor cultures, tripartite predatory/prey cultures, sub-lethal antibiotics) will be presented to myxobacteria (domesticated and cave-isolated) and the resultant metabolome will be evaluated by mass spectrometry. Select features will be targeted for isolation based on divergence from known myxobacterial NPs (via molecular networking and ion mobility analyses). Structural elucidation will be accomplished via column chromatography, tandem mass spectrometry, and NMR. In Aim 2, myxobacterial metabolomes and metabolites will be assessed for their role in mammalian chemical biology using multiplexed activity metabolomics (MAM), a high-throughput technique that enables the simultaneous evaluation of a large number of metabolites against an array of cell subtypes and cellular pathways. MAM incubates fractionated metabolites against a cancer-derived cell line and assays them against 8-15 fluorescent antibodies targeting cellular function (e.g. apoptosis, DNA damage, and viability). Cytometric analysis and correlation of the resulting spectral and bioactivity chromatograms prioritizes leads to test against primary human cancer cells and to structurally decipher (Aim 1). !